Structural analysis of covalent peptide dimers, bis(pyridine-2-carboxamidonetropsin)(CH_2)_(3-6), in complex with 5'-TGACT-3' sites by two-dimensional NMR

The peptide pyridine-2-carboxamidonetropsin (2-PyN) binds specifically in the minor groove of 5'-(A,T)G(A,T)C(A,T)-3' sequences as a side-by-side antiparallel dimer. Tethering two 2-PyN ligands through the nitrogens of the central pyrrole rings with propyl, butyl, pentyl and hexyl linkers affords covalent peptide dimers, bis(pyridine-2-carboxamide-netropsin)(CH_2)_(3-6), which bind in the minor groove of DNA with increased binding affinities and improved sequence specificities. Two-dimensional NMR studies of the complexes formed upon binding of these covalent peptide dimers to an oligonucleotide containing a 5'-TGACT-3' site reveal that the dimeric peptides bind as intramolecular dimers with nearly identical geometry and peptide-DNA contacts as in the (2-PyN)_2•5'-TGACT-3' complex

NMR structure of a cyclic polyamide-DNA complex

The solution structure of a cyclic polyamide ligand complexed to a DNA oligomer, derived from NMR restrained molecular mechanics, is presented. The polyamide, cyclo-gamma-ImPyPy-gamma-PyPyPy-, binds to target DNA with a nanomolar dissociation constant as characterized by quantitative footprinting previously reported. 2D (1)H NMR data were used to generate distance restraints defining the structure of this cyclic polyamide with the DNA duplex d(5'-GCCTGTTAGCG-3'):d(5'-CGCTAACAGGC-3'). Data interpretation used complete relaxation matrix analysis of the NOESY cross-peak intensities with the program MARDIGRAS. The NMR-based distance restraints (276 total) were applied in restrained molecular dynamics calculations using a solvent model, yielding structures with an rmsd for the ligand and binding site of approximately 1 A. The resulting structures indicate some distortion of the DNA in the binding site. The constraints from cyclization lead to altered stacking of the rings in the halves of the cyclic ligand relative to unlinked complexes. Despite this, the interactions with DNA are very similar to what has been found in unlinked complexes. Measurements of ligand amide and DNA imino proton exchange rates indicate very slow dissociation of the ligand and show that the DNA can undergo opening fluctuations while the ligand is bound although the presence of the ligand decreases their frequency relative to the free DNA

NMR structure of a cyclic polyamide-DNA complex

The solution structure of a cyclic polyamide ligand complexed to a DNA oligomer, derived from NMR restrained molecular mechanics, is presented. The polyamide, cyclo-gamma-ImPyPy-gamma-PyPyPy-, binds to target DNA with a nanomolar dissociation constant as characterized by quantitative footprinting previously reported. 2D (1)H NMR data were used to generate distance restraints defining the structure of this cyclic polyamide with the DNA duplex d(5'-GCCTGTTAGCG-3'):d(5'-CGCTAACAGGC-3'). Data interpretation used complete relaxation matrix analysis of the NOESY cross-peak intensities with the program MARDIGRAS. The NMR-based distance restraints (276 total) were applied in restrained molecular dynamics calculations using a solvent model, yielding structures with an rmsd for the ligand and binding site of approximately 1 A. The resulting structures indicate some distortion of the DNA in the binding site. The constraints from cyclization lead to altered stacking of the rings in the halves of the cyclic ligand relative to unlinked complexes. Despite this, the interactions with DNA are very similar to what has been found in unlinked complexes. Measurements of ligand amide and DNA imino proton exchange rates indicate very slow dissociation of the ligand and show that the DNA can undergo opening fluctuations while the ligand is bound although the presence of the ligand decreases their frequency relative to the free DNA

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry

Many candidate unnatural DNA base pairs have been developed, but some of the best-replicated pairs adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replication for one such pair,dNaM-d5SICS, and show that efficient replication results from the polymerase itself, inducing the required natural-like structure

Efforts Toward Expansion of the Genetic Alphabet: Structure and Replication of Unnatural Base Pairs

Expansion of the genetic alphabet has been a long time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semi-synthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogs, such as propinyl isocarbostyril (d PICS ), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogs inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogs that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluoro benzene (d 3FB ), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self pair formed between two d 3FB analogs is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d 3FB or d PICS self pairs. We find that the large aromatic rings of d PICS pair in an intercalative manner within duplex DNA, while the d 3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl substituted derivatives of d 3FB (d 5Me3FB ) paired opposite d 3FB or the unsubstituted analog (d BEN ). In all, the data suggest that structure, electrostatics and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated